Beating the diffraction-limit using CARS microscopy

I get to blow my own horn about some of our research. We have some …

Every now and again, I look at the work of other scientists and feel envious. Why haven't I managed to do something that exciting? Some people will respond by trying to find a new angle on the the novel work. But others will ask "How can we apply such wonderful ideas in a totally new fashion?" This is the approach our research group has taken with stimulated emission depletion (STED) microscopy.

STED is a wonderful technique that produces fantastically detailed images of living cells that have been tagged with fluorescent molecules. We wondered, "Why can't you use STED-like processes for label-free microscopy?" In trying to answer that question, we discovered that it might well be possible.

STED and CARS

First, a quick refresher on STED microscopy. In STED, the sample is stained with a fluorescent dye that (hopefully) attaches only to the parts of the cell that you are interested in looking at. When illuminated by a laser, these dyes absorb the laser light and then emit light at a slightly longer wavelength. This provides a beautiful, high-contrast image. Unfortunately, it can only resolve features at about the scale of the wavelength of the excitation light. STED makes use of the fact that where the laser has excited the dye, a population inversion exists—most of the dye is in the excited state and ready to emit.

A second laser with a different wavelength causes all the dye to emit through stimulated emission—the same process that provides the light for a laser. This second laser has a beam with a hole in the middle, called a donut beam. As a result, the excited state population is eliminated everywhere except at the very center of the beam. Any emission after this is known to come from that (very small) remaining volume that was in the donut hole, which allows very high resolution images.

We decided to take this general principle and see if it could be applied a coherent anti-Stokes Raman scattering (CARS) microscope. Which means it's time for a refresher on the workings of CARS. The first thing to note is that CARS involves Raman interactions, where the light fields are pretty much transparent to the sample. Instead, a tiny fraction of the photons scatter off a molecule in such a way that they set the molecule vibrating. In doing so, the photons lose a small amount of energy, changing their wavelength. The vibrational motion of the molecule can be figured out by examining how much energy was lost during the scattering.

This process is generally very inefficient, and the Raman scattered photons go everywhere. CARS provides a second light field that just happens to have the wavelength of the scattered photons. This drives the ground and the vibrational states of the molecules to change in a synchronized fashion.

The molecules in the vibrational state are then strongly driven back into the ground state. They do this by coupling to the light fields and emitting photons that have a higher energy than the original photons. The nice thing about this process is that it is more efficient than Raman scattering and, because it is coherent, a good microscope can collect more of these higher-energy photons.

In terms of microscopy, we like CARS because you label samples by the unique combinations of their natural vibrational modes, which means that you don't need to stain your samples with fluorescent dyes. But I think, given the choice, a biologist would rather have the increased resolution of STED than the advantages of label-free microscopy. And, in an ideal world, they would rather have both.

Best of both worlds

Applying STED principles to CARS is not straightforward, though, because there are only ever a few molecules in the excited vibrational state. Since there is no population inversion, we cannot use a laser to drive population back to the ground state. The thing that turns out to be important is the coherence. This synchronicity between the ground state and the vibrational state is the process that allows CARS to proceed—without it, there is no CARS emission. The key, then, is to destroy or prevent the build-up of the coherence.

It turns out that this can be done under some circumstances. Before the light fields used to generate CARS are turned on, we blast the sample with a large amount of radiation that is resonant with some other vibrational mode. This excites vibrations in a large fraction of the molecular population. However, these vibrations, through their very motion, excite other vibrational modes. If we hit the sample with enough light, we end up with the population spread evenly over a large number of vibrational modes, including the one that we want to look at with CARS.

What does this get us? Well, it turns out that these coherent changes in vibrational motion require that the ground and vibrational state populations are unequal. By hitting the molecules with enough light, we spread the population over many vibrational states and, eventually, the populations equalize. Once equalized, the CARS lasers fail to establish coherence, and no signal emerges.

The nice thing about this is that we can basically apply all the lessons learned in the development of STED—using a donut shaped beam limits CARS emissions to a tiny volume in the center. On a slightly more problematic note, we need to use lasers that excite vibrational states—in other words these laser have to emit light with wavelengths longer than 2.4 micrometers. These light sources tend to be a bit, um, cumbersome. Also, the high-quality optics required for a microscopy setup are not as readily available.

Another concern is the amount of laser power required to generate equal populations. In this case, we have some good news. If it can be done, it will be done at powers that will not damage the sample. This is because the process doesn't have to take place rapidly, so one can stretch the pulse out to lower the power. On the other hand, the energy is still going somewhere, so we are kind of hoping that it dissipates harmlessly.

Where, you might ask, is the experiment? Well, we are currently looking for a new student to put on the problem, so hopefully it won't be too far away.

aiken_d: STED has been shown to improve photolithography. But the mask patterns are rather complicated (since you need an inverse and then around every feature you need STED to reduce the size of the feature--so the sted beam needs to have a node that follows the shapes of the features.

So, how does STED improve lithography? What sort of dye based mask is being used? computational lithography is similar (though a completely independent technique), and local-field approaches are also concetually similar, but I can't see how anything that is really STED is lithography.

In any case, I was actually curious why you couldnt use a OPA+ a high power pulsed (fs) laser, and mix down to 2.4ish um. That is not so hard. Usually, frequency mixing is done to generate a higher energy, but for every upconverted photon there is a down. Are techniuqes like that used in the field?

Chris Lee / Chris writes for Ars Technica's science section. A physicist by day and science writer by night, he specializes in quantum physics and optics. He lives and works in Eindhoven, the Netherlands.